Characterization of upFc, a fragment of human immunoglobulin G1 produced by pepsin in urea

Abstract

The digestion of human IgG1[immunoglobulin G1]/.kappa. myeloma proteins with pepsin in the presence of 8 M-urea produces fragments that differ from those produced by aqueous peptic digestion, and from other characteristic Ig fragments. Fb''2, the larger urea/pepsin fragment, was previously shown to consist of the constant regions of the L chains, and the CH1 [H chain constant region 1] domains and hinge regions of the H chains. The smaller fragment, upFc, was characterized in this paper. After reduction, 3 peptides were released from fragment upFc. Amino acid sequencing, N- and C-terminal determinations and amino acid compositions have enabled these peptides to be identified as residues Ile-253 to Leu-306, residues Thr-307 to Asp-376 and residues Thr-411 to Gly-446 of the H chain. Fragment upFc contains the entire Fc region, beginning at residue Ile-253, except for a 34-residue section from within the CH3-domain disulfide loop. Peptic digestion of IgG1/.kappa. proteins in 8 M-urea provides a method for isolating from .gamma.1 H chains 5 homogeneous peptides in good yield, which account for almost the entire constant region. Characterization of fragments Fb''2 and upFc has shown that the action of pepsin in urea is entirely different from that of aqueous pepsin. Two .gamma.1 H chains differed in sequence at 3 positions from the sequence reported for protein Eu.